PROTEIN EXPRESSION AND PURIFICATION 1, 77-80 (1990) Purification of a Membrane-Associated Serine Esterase from Murine Cytotoxic T Lymphocytes by a Single Reverse-Phase Column Shibo Jiang,’ Cynthia S. Hasselkus-Light, David M. Ojcius, and John Ding-E Young Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Avenue, New York, New York 10021 Received May 10, 1990, and in revised form July 12, 1990 The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-RS, were isolated by subcellu- lar fractionation. After dissolving in detergent-con- taining buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N”-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the frac- tion with highest serine esterase activity. Under so- dium dodecyl sulfate-polyacrylamide gel electrophore- sis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP- 3lgranzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase col- umn method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further charac- terized for function. 0 1990 Academic PRSS, IN. A serine esterase (SE)’ activity which hydrolyses W- benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) had i Present address: Biochemical Virology Laboratory, The Lindsley F. Kimball Research Institute of New York Blood Center, 310 East 67 Street, New York, NY 10021. ’ Abbreviations used: ADCC, antibody-dependent cellular cytotox- icity; BLT, N”-benzyloxycabonyl-L-lysine thiobenzyl ester; CAPS, 3-(cyclohexylamino)-l-propanesulfonic acid; CTL(s), cytotoxic T lymphocyte(s); DFP, diisopropylfluorophosphate; DTT, dithiothrei- tol; LAK, lymphokine-activated killer; MCSP-3, mouse CTL serine protease-3; PME, 2-mercaptoethanol; NK, natural killer; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SE(s), serine esterase(s); TCR, T cell receptor; TFA, trifluoroacetic acid. 1046.5928/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. been identified in cytotoxic T lymphocytes (CTLs) and was originally described as CTL specific (1). Thus, this trypsin-like protease was previously proposed as a marker for CTL (1). Later, a family of SEs was isolated through subtractive hybridization cloning and conven- tional biochemical techniques from CTL and NK cyto- plasmic granules (2-6). Through a combination of the techniques of immunochemistry and amino acid and cDNA sequencing, it has been demonstrated that the overall structures of these SEs, designated as gran- zymes (9), are homologous and some of their domains are highly conserved (7-9). The function of these SEs remains speculative at the present time even though some indirect evidence sug- gests that SEs may participate in cell-mediated killing since various protease inhibitors, such as diisopropyl- fluorophosphate (DFP) and phenylmethylsulfonyl fluo- ride, are capable of blocking killing mediated by CTLs (lo), NK cells (ll), and lymphocytes engaged in ADCC (12). However, the fact that no direct cytotoxicity me- diated by SEs has been detected indicates that these enzymes may only have an intermediate processing function that may be required to lyse targets by other lytic mediators (13). Most of these SEs were isolated from the cytoplasmic granules of cytolytic lymphocytes (l-3,14) and some from the supernatant of CTLs which were specifically stimulated via the TCR (1516). In the present study, we describe for the first time the isolation of a SE from the plasma and organelle membranes of a CTL line. Analy- sis of the amino-terminal sequence shows that this SE is identical to granzyme F, which is also known as MCSP- 3 (7). The function of this SE in CTLs is still under investigation. MATERIALS AND METHODS Cells. The murine cytotoxic T lymphocyte line, CTLL-R8, was maintained in aMEM (GIBCO, Grand 77